The present invention relates to a method of preparing a monoclonal antibody against a surface antigen, and in particular such a surface antigen which constitutes only an insignificant amount of the total antigen injected in a mammal and a surface antigen which easily loses its in vivo conformation. The problem of an insignificant amount of antigen relative to the total amount of antigen for example sets in when the antigen material used to inject a mammal is derived from another mammalian species.
The problems associated with the antigens specified above become clear when, for example, it is desired to make monoclonal antibodies against the specificity determining part of a T-cell receptor (TCR). Such (monoclonal) antibodies are known as anti-clonotype antibodies as they recognize the antigen-specific part (or clonotype structure) of a T-cell receptor of one particular T-cell clone. While (murine) monoclonal anti-clonotype antibodies against murine T-cell receptors (TCR) have been reported only occasionally, there are even less (murine) monoclonal anti-clonotype antibodies known against human T-cell receptors. T-cells are hard to culture, making it difficult to obtain and purify a sufficient amount of antigen, i.e. T-cell receptor protein. This in turn makes it difficult to obtain an immune response and also makes screening difficult. In one of the few cases where a monoclonal anti-clonotype antibody is available against a human TCR, these problems did not arise because the antigen was present on Jurkat leukaemia cells (ref. 1). As leukaemia cells can be cultured in unlimited amounts, the problem of a shortage of antigen did not arise. Therefore, until now no method was available to prepare monoclonal antibodies against rare and/or very unstable antigens without having to screen unfavourably large numbers of hybridoma clones.
The object of the present invention is to provide a method of preparing a monoclonal antibody against a cell surface antigen under unfavourable circumstances as described above, said method dramatically reducing the number of hybridoma clones to be screened, rendering the method according to the invention more efficient or even successful where methods according to the state of the art fail.
To this end the method according to the invention comprises the steps of
1) injecting a mammal with cell surface antigen-comprising material, said material being chosen from the group consisting of i) whole cells and ii) a membrane fraction obtained by treating whole cells;
2) isolating a B-cells-containing cell fraction from the spleen of said mammal;
3) enriching said cell fraction obtained in step 2 in B-cells specific for said cell surface antigen by contacting the cell fraction with carrier-bound material of cells related to said whole cells, said carrier-bound related material of cells lacking said cell surface antigen, and separating B-cells bound to carrier-bound material of related cells from the enriched unbound B-cells-containing cell fraction to be used in the next step;
4) subjecting the enriched B-cells-containing cell fraction obtained in the previous step to limiting dilution followed by clonal expansion;
5) selecting a B-cell clone and immortalising said selected B-cell clone using a small-scale fusion technique; and
6) selecting and cloning a hybridoma capable of producing antibodies which specifically bind said cell surface antigen, followed by isolating a monoclonal antibody-comprising fraction from supernatant of said hybridoma.
For the purpose of keeping the description and the claims readable and intelligible, here the term cell is used not only to indicate mammalian cells but also viruses, and in particular membrane-comprising viruses.
The term carrier-bound material of cells encompasses intact or whole cells (and viruses), membrane fractions of said intact whole cells (and viruses), or substantially purified surface-antigens or complexes thereof.
Unless specified otherwise, the term whole cell refers to cells having the surface antigen of interest. The term related cells refers to cells which preferably differ only in that they lack the surface-antigen of interest, or more specifically at least lack the immunological determinant against which it is desired to obtain a monoclonal antibody.
Surprisingly it proved to be possible to enrich the cell fraction in B-cells specific for the cell surface antigen without the use of cell surface antigen by contacting the isolated B-cells-containing cell fraction with material of cells from the same species as the cell surface antigen-comprising material was derived of but lacking the cell surface antigen. In other words, by contacting the B-cells with irrelevant related cells or cell material and removing bound B-cells an enrichment was achieved.
Thus it is possible to prepare a monoclonal antibody against a minor cell surface antigen, even if said cell surface antigen suffers from conformational instability.
European patent EP 0 488 470 and ref. 3, which is based on this European patent, describe a method in which I) a mammal is injected with an antigen, II) a B-cells-containing fraction is isolated, III) B-cells specific for the antigen are selected, IV) the enriched fraction is subjected to clonal expansion and after V) selecting a B-cell clone and immortalisation using a small-scale fusion technique a VI) hybridoma is selected and cloned followed by isolation of a monoclonal antibody comprising fraction. In step III the B-cells are selected by binding them on an antigen coated plastic surface or by resetting them with antigen coated paramagnetic beads. The non-specific B-cells are removed by washing. Thus, apart from other differences, this method relies on the ample availability and use of antigenxe2x80x94during the selection 2 xcexcg/ml antigen is used to coat plates with antigenxe2x80x94, whereas the present invention solves the problem for a case in which antigen is available in very limited amounts and even highly impure.
A preferred embodiment is characterized in that the mammal injected with surface antigen is of a different species than the mammalian species from which the surface antigen originates.
The method according to the present invention is very suitable for preparing monoclonal antibodies when many antigens capable of eliciting an immuneresponse are present.
This situation exists in particular when the surface antigen has a constant section and a variable section, wherein at least a part of said variable section defines a specificity determining part of said surface antigen.
According to a preferred embodiment receptor molecule-comprising material is used as the cell surface antigen-comprising material.
The method according to the invention is particularly suitable for preparing monoclonal antibodies against the specificity determining part of a receptor molecule. The specificity determining part of the receptor molecule is only a minor part of the receptor molecule, which in itself is a minor constituent of all the moleculesxe2x80x94and thus antigensxe2x80x94on the surface of a cell.
A preferred target according to the invention is a T-cell clone, which T-cell clone is used to prepare the receptor molecule-comprising material.
A T-cell may be used as a whole cell, or used for the preparation of a membrane fraction thereof. Thus, in the former case the term prepare may, here, relate to the mere isolation of a T-cell or resuspending in a different medium.
According to preferred embodiments the membrane fraction in step 1 is obtained by mechanical treatment of the whole cells, and the cell surface antigen-comprising material is injected in the mammal in the absence of an adjuvant.
Both measures help to prevent loss of the in vivo conformation of the surface antigen.
Furthermore, the enriched B-cells-containing cell fraction is preferably further enriched (step 3) by contacting the cell fraction with carrier-bound cell surface antigen comprising material chosen from the group of i) whole cells, ii) a membrane fraction obtained from said whole cells, and iii) substantially purified cell surface antigen, and subsequently separating B-cells not bound to said carrier-bound material from B-cells bound to said carrier-bound material, said B-cells bound to said carrier-bound material comprising the further enriched cell fraction.
This further enrichment can be designated as a positive selection technique, selecting specifically specific B-cells. Thus, though hardly any antigen is available, further enrichment could be achieved.
According to a preferred embodiment paramagnetic beads are advantageously used as the carrier.
The use of paramagnetic beads during the enrichment facilitates the separation of wanted and unwanted B-cells.
An insignificant amount of antigen also presents a problem during screening. According to a preferred embodiment, the selection in at least one of the steps 5 and 6 is conducted using an agglutination assay wherein supernatant of the B-cell clone is contacted with a carrier coated with antibodies capable of binding antibodies of the species of the injected mammal used in step 1 and whole cells bearing the cell surface antigen, and agglutination is detected.
Simply mixing B-cell culture supernatant, whole cells and a carrier coated with antibodies capable of binding antibodies of the mammalian species used in step 1, allows for a very sensitive and rapid detection of suitable clones while washing is not required.
Advantageously related cells (whole cells which lack the surface antigen) are used as controls.
This allows for the rejection of false-positive clones, and saves time by avoiding superfluous small-scale fusions.
According to a preferred embodiment of the method according to the invention, the selected B-cell clone in step 5 is mixed with myeloma cells and subjected to mini-electrofusion.
Mini-electrofusion allows for the efficient fusion of very small numbers (e.g. hundreds) of cells.
The invention also relates to a pharmaceutical composition comprising a monoclonal antibody prepared according to the invention admixed with a suitable excipient.
Furthermore the present invention relates to a monoclonal antibody reactive with the clonotypic structure of a T-cell receptor.
Such monoclonal antibody can be used for diagnostic purposes as well as for the preparation of a pharmaceutical composition.
More specifically the T-cell receptor is a T-cell receptor associated with an auto-immune disease, and in particular rheumatoid arthritis.
Preferably the monoclonal antibody is reactive with the T-cell receptor of a HC gp-39 reactive T-cell clone, and in particular with the T-cell clone H.243 (ECACC accession No. 96103122).
Specific examples of suitable monoclonal antibodies are those produced by a hybridoma chosen from the group consisting of TCR 69 (ECACC accession No. 96103118), TCR 70 (ECACC accession No. 96103119), TCR 72 (ECACC accession No. 96103120) and TCR 83 (ECACC accession No. 96103121).
The above deposits were made under the Budapest Treaty on Oct. 31, 1996 with the European Collection of Cell Cultures, Centre for Applied Microbiology and Research, Porton Down, Salisbury SP4 0J6, United Kingdom.
As indicated above, the invention also relates to pharmaceutical composition comprising a monoclonal antibody according to the invention admixed with a suitable excipient, suitable for the treatment of rheumatoid arthritis.
Finally the invention relates to the diagnostic use of a monoclonal antibody chosen from the group consisting of a monoclonal antibody prepared using the method according to the invention and a monoclonal antibody according to the invention. Also a diagnostic reagent comprising the antibody is an embodiment of the invention.
Now the invention will be explained in further detail with reference to the following example, said example showing the best mode of carrying out the invention applied to the preparation of a murine monoclonal antibody specific for a clonotype of a human T-cell receptor.